Kun:LabNotes/ExonomeSeq/2008-1-30

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Exp.1 Library construction on the Cosmic3760 exons from NA10835 and NA20431 (01/27/2008)

Mme I digestion on the PCR amplicon

 Mix pooled 50ul amplicons from two tubes, add 1ul 32mM SAM, 4ul Mme I.
 37C 2h -> 75C 20min.
 PAGE size selection of 50ul digestions.
 Split the purified DNA into two tubes, one for USER digestion and the other for SolN6 primed synthesis.

USER enzyme digestion

Add 1ul USER enzyme into 10ul of DNA in ddH2O -> 37C 10min.

End repair

Set up end repair reaction (with NEB quick blunting kit)

                 USER         
     DNA         10ul        
     10X buffer 1.5ul        
     1mM dNTP   1.5ul         
     Enzyme mix 0.5ul
     H2O        1.5ul          
     RT for 30min -> 75C 10min

Ligation

      DNA                  15ul              
  50uM Solexa_1             2ul              
  50uM Solexa_2             2ul               
  2X Quick ligation buffer 20ul               
  Quick ligase              1ul
  mix well -> room temperature for 20min 
  Purified with MinElute columns.

Nick-translation

  DNA                    10ul
  10X ThermoPol Buffer  1.2ul
  10mM dNTP             0.3ul
  8U/ul Bst pol         0.5ul
  65C 25min -> place on ice

SolN6 primed synthesis

                       SolF/Rn6     
     DNA                 10ul      
     10X Klenow buffer    5ul     
     100uM SolFn6         1ul
     10mM dNTP            2ul      
     H2O                 30ul      

94C 2min -> 16C 1min -> add 1ul 20U/ul Klenow exo- -> 16C 10min -> 37C 10min -> Clean up the reaction with G25 column, then add 6 ul 10X buffer, 2ul dNTp, 1ul 100uM SolRn6-> 94C 1min -> 16C 1min -> add 1ul 20U/ul Klenow exo-, 1ul T4 DNA pol-> 16C 10min -> 37C 10min -> purified with MinElute columns, eluted with 10ul EB.

PCR

    Nick-translated DNA      5ul       SolN6 primed DNA  5ul
    2X iProof master mix    50ul                        50ul
    100uM Solexa_PCR_up   0.25ul                      0.25ul
    100uM Solexa_PCR_lo   0.25ul                      0.25ul
    H2O                     44ul                        44ul
    50X SYBG I             0.4ul                       0.4ul
    98C 30sec -> 18 cycles of (98C 10sec -> 65C 20 sec -> 68C 30sec) -> 72C 3min ->15C hold
 File:2008-02-01-SolLib-PCR.png
 
 
 I decided to do PAGE purification of the remaining 10ul PCR templates, then do PCR.
 File:2008-02-01-PAGE.png
 After shearing the gel, I directly added 100ul 1X iProof mix with 0.2X SYBR Green I but no primer.
 Incubate at thermal cycler at 37C for 1h, then start thermal cycling with same protocol.
 However, I realized after 15 cycles that I forgot to added the primers. 
 So I took the tubes out, add Solexa_PCR_lo/up primers (0.4uM), and repeat 18 cycles of thermal cycling.
 I took tube A/B out, and did 5 more cycles on C/D.
 PAGE purification on the A/B amplicons from PAGE purified templates, and C/D amplicons from unpurified templates.
 The amplification A/B didn't work.
 File:2008-02-01-SolLib-PCR.png